Retinoid isomerase, RPE65, and Lecithin-retinol acyltransferase (LRAT) are important in recycling 11-cis- retinal in retinal pigment epithelium (RPE). Mutations in either gene lead to Leber congenital amaurosis (LCA), an inherited retinal degenerative disease characterized by severe loss of vision in childhood and early degeneration of cones followed by rods. The main pathologic features are recapitulated by two models, Rpe65-/- and Lrat-/- in which 11-cis-retinal is lacking. However, the mechanisms responsible for early cone degeneration in both mouse models and human patients are not well understood. Specifically, it is unclear why ventral and central cones in mouse models die much more rapidly than dorsal cones. Similarly, it is unclear why blue cone function is lost early in patients. The objective of this application is to define the mechanism responsible for the rapid cone degeneration in LCA using the Lrat-/- model. Our central hypothesis is that cone opsin determines the rate of cone photoreceptor degeneration in LCA. We formulated this hypothesis based on our preliminary data showing the short-wavelength (SW) opsins are prone to aggregation/accumulation triggering endoplasmic reticulum (ER) stress than the medium/long-wavelength opsin. In Aim 1, we will dissect the relative contributions of S and M opsin to cone degeneration in Lrat-/- mice by genetically deleting S-opsin or M-opsin. In Aim 2, we will determine the structural basis on why chromophore deprivation causes S-opsin but not M-opsin aggregation. We identified a phenylalanine-rich region in the short-wavelength opsin family (SW1) but absent from the medium/long-wavelength opsin family. Since aromatic residues play a significant role in the aggregation of proteins by directing self-assembly via - interactions, we hypothesize that the phenylalanine-rich region in the S-opsin is responsible for its aggregation in Lrat-/- cones. We designed two sub-aims to test this hypothesis. Sub-aim 2A; swap the phenylalanine-rich region in the S-opsin (70-125) with the homologous region of M-opsin (86-141) by gene targeting. Sub-aim 2B, inhibit S-opsin aggregation with N- (1, 4-dihydro-1, 4-dioxo-2-naphthalenyl)-L-tryptophan (NQTrp) by disrupting the - interactions between the phenylalanine-rich region. We expect these studies will define the mechanism on why S-cones degenerate faster than M-cones in RPE65/LRAT-LCA. PUBLIC HEALTH RELEVANCE: These studies will define the mechanism on why S-cones degenerate faster than M-cones in RPE65/LRAT- LCA. The proposed mechanism that cone opsin aggregation is responsible for rapid cone degeneration can be exploited in designing novel therapeutic agents in the treatment of LCA. For example, NQTrp has the potential to serve as a lead for developing a new class of therapeutic drugs for LCA by blocking cone opsin aggregation.